Signalling by immunoreceptors is orchestrated at specific plasma membrane microdomains, referred to as lipid rafts. Here we present a proteomics approach to the temporal analysis of protein association with lipid rafts following T-cell antigen receptor (TCR) triggering. We show that TCR engagement promotes the temporally regulated recruitment of proteins participating in the TCR signalling cascade to lipid rafts. Furthermore, TCR triggering results in profound modifications in the composition of lipid rafts involving a number of proteins associated either directly or indirectly with both plasma and intracellular membranes. Raft-associated proteins can be clustered according to their temporal profile of raft association. The data identify lipid rafts as highly dynamic structures and reveal a dramatic impact of surface TCR triggering not only on components of the TCR signalling machinery but also on proteins implicated in a number of diverse cellular processes.
Abbreviations used: ARF, ADP ribosylation factor; 1D, one-dimensional; 2D, two-dimensional; ERK, extracellular signal-regulated protein kinase; GM130, Golgi matrix protein 130; GPI, glycosylphosphatidylinositol; LAT, linker of activated T-cells; MALDI-TOF, matrix-assisted laser-desorption ionization—time-of-flight; PLC, phospholipase C; SLP-76, SH2-domain-containing leucocyte protein of 76kDa; TCR, T-cell antigen receptor; VDAC, voltage-dependent anion channel; ZAP-70, zeta-associated protein of 70kDa.
These authors contributed equally to this work.